The localization of PHRAGMOPLAST ORIENTING KINESIN1 at the division site depends on the microtubule-binding proteins TANGLED1 and AUXIN-INDUCED IN ROOT CULTURES9 in Arabidopsis

Abstract Proper plant growth and development require spatial coordination of cell divisions. Two unrelated microtubule-binding proteins, TANGLED1 (TAN1) and AUXIN-INDUCED IN ROOT CULTURES9 (AIR9), are together required for normal growth and division plane orientation in Arabidopsis (Arabidopsis thaliana). The tan1 air9 double mutant has synthetic growth and division plane orientation defects, while single mutants lack obvious defects. Here we show that the division site-localized protein, PHRAGMOPLAST ORIENTING KINESIN1 (POK1), was aberrantly lost from the division site during metaphase and telophase in the tan1 air9 mutant. Since TAN1 and POK1 interact via the first 132 amino acids of TAN1 (TAN11–132), we assessed the localization and function of TAN11–132 in the tan1 air9 double mutant. TAN11–132 rescued tan1 air9 mutant phenotypes and localized to the division site during telophase. However, replacing six amino-acid residues within TAN11–132, which disrupted the POK1–TAN1 interaction in the yeast-two-hybrid system, caused loss of both rescue and division site localization of TAN11–132 in the tan1 air9 mutant. Full-length TAN1 with the same alanine substitutions had defects in phragmoplast guidance and reduced TAN1 and POK1 localization at the division site but rescued most tan1 air9 mutant phenotypes. Together, these data suggest that TAN1 and AIR9 are required for POK1 localization, and yet unknown proteins may stabilize TAN1–POK1 interactions.

. Positioning and construction of a new cell wall (cell plate) during cytokinesis involves two microtubule-and microfilament-rich cytoskeletal structures: the preprophase band (PPB) and the phragmoplast, respectively (Smertenko et al., 2017). The PPB is a ring of microtubules, microfilaments, and proteins that forms at the cell cortex just beneath the plasma membrane during G2; this region is defined as the cortical division zone (Van Damme, 2009;Li et al., 2015;Smertenko et al., 2017). The cortical division zone is characterized by active endocytosis mediated by TPLATE-clathrin-coated vesicles that may deplete actin and the actin-binding kinesin-like protein KCA1/KAC1 (Hoshino et al., 2003;Vanstraelen et al., 2004;Panteris, 2008;Karahara et al., 2009;Suetsugu et al., 2010;Kojo et al., 2013). After nuclear envelope breakdown, the PPB disassembles and the metaphase spindle, an antiparallel microtubule array with its plus-ends directed toward the middle of the cell, forms (Dixit and Cyr, 2002). After the chromosomes are separated, the phragmoplast is constructed from spindle remnants to form another antiparallel array of microtubules (Lee and Liu, 2019). The phragmoplast microtubules are tracks for the movement of vesicles containing cell wall materials toward the forming cell plate (McMichael and Bednarek, 2013;Müller and Jürgens, 2016). The phragmoplast expands by nucleation of new microtubules on preexisting microtubules (Murata et al., 2013;Smertenko et al., 2018) and is partially dependent on the mitotic microtubule-binding protein ENDOSPERM DEFECTIVE1 and the augmin complex to recruit gamma tubulin to phragmoplast microtubules (Nakaoka et al., 2012;Lee et al., 2017). Finally, the phragmoplast reaches the cell cortex, and the cell plate and associated membranes fuse with the mother cell membranes at the cell plate fusion site previously specified by the PPB (van Oostende-Triplet et al., 2017). TANGLED1 (TAN1, AT3G05330) was the first protein identified to localize to the plant division site throughout mitosis and cytokinesis (Walker et al., 2007). In maize (Zea mays), the tan1 mutant has defects in division plane orientation caused by phragmoplast guidance defects (Cleary and Smith, 1998;Martinez et al., 2017). TAN1 bundles and crosslinks microtubules in vitro (Martinez et al., 2020). In vivo, TAN1 promotes microtubule pausing at the division site (Bellinger et al., 2021). TAN1, together with other division site-localized proteins, is critical for the organization of an array of cell cortex-localized microtubules that are independent from the phragmoplast. These cortical-telophase microtubules accumulate at the cell cortex during telophase and are subsequently incorporated into the phragmoplast to direct its movement toward the division site (Bellinger et al., 2021).
Other important division site-localized proteins were identified through their interaction with TAN1, such as the division site-localized kinesin-12 proteins PHRAGMOPLAST ORIENTING KINESIN (POK1) and POK2 (Müller et al., 2006;Lipka et al., 2014). Similar to PHRAGMOPLAST-ASSOCIATED KINESIN-RELATED PROTEIN (PAKRP1) and PAKRPL1 (Pan et al., 2004;Lee et al., 2007), which are other kinesin-12 proteins, POK2 localizes to the phragmoplast midline during telophase and plays a unique role in phragmoplast expansion . Together, POK1 and POK2 are required to guide the phragmoplast to the division site (Müller et al., 2006;Herrmann et al., 2018). The pok1 pok2 double mutant of Arabidopsis (Arabidopsis thaliana) has stunted growth and misplaced cell walls as a result of phragmoplast guidance defects (Müller et al., 2006). The pok1 pok2 double

IN A NUTSHELL
Background: Unlike animal cells, plant cells cannot move due to their semi-rigid cell walls. The correct positioning of the new cell wall is important for overall plant growth and development. Three microtubule-binding proteins are involved in division plane orientation: TANGLED1 (TAN1), AUXIN-INDUCED IN ROOT CULTURES9 (AIR9), and PHRAGMOPLAST ORIENTING KINESIN1 (POK1). These proteins localize as a ring at the edge of the cell where the new cell wall will insert during cell division, at a position called the division site. Here, we focused on how TAN1 and POK1 interactions promote their localization to the division site, and their function in plant growth and division plane positioning.
Question: How do TAN1 and AIR9 contribute to POK1 localization, and how does POK1 localization affect new cell wall placement?
Findings: TAN1 and AIR9 together maintain POK1 at the division site in Arabidopsis thaliana. When mutant versions of TAN1 that no longer interact with POK1 were transformed into the tan1 air9 double mutant, POK1 and TAN1 localization was partially disrupted and cell wall placement defects occurred. This suggests that POK1 interaction with TAN1 is important for their correct division site localization and new cell wall placement.
Next steps: This work strongly suggests that yet unknown proteins mediate TAN1 and POK1 interaction. Discovering what those proteins are, and how AIR9 contributes to division plane positioning are next. Understanding how plants position their division plane will contribute to understanding plant growth and has the long-term potential to contribute to next-generation crop development. mutant also fails to maintain TAN1 at the division site after entry into metaphase (Lipka et al., 2014). This suggests that TAN1 maintenance at the division site after metaphase is dependent on POK1 and POK2.
In Arabidopsis, the tan1 mutant has minor phenotypic differences compared with wild-type (WT) plants (Walker et al., 2007). However, the tan1 auxin-induced-in-root-cultures9 (air9) double mutant, which has no obvious defects (Buschmann et al., 2015), resulted in a synthetic phenotype consisting of reduced root growth, increased root cell file rotation, and phragmoplast guidance defects (Mir et al., 2018). TAN1 and AIR9 are unrelated microtubulebinding proteins that both localize to the division site (Buschmann et al., 2006;Walker et al., 2007). Both TAN1 and AIR9 colocalize with the PPB. TAN1 remains at the division site throughout cell division, while AIR9 is lost from the division site upon PPB disassembly and then reappears at the division site during cytokinesis when the phragmoplast contacts the cortex. When full-length TAN1 fused to YELLOW FLUORESCENT PROTEIN (TAN1-YFP) and driven either by the constitutive viral cauliflower mosaic CaMV35S promoter (p35S:TAN1-YFP) or the native promoter with the fluorescent protein as either an N-or C-terminal fusion (pTAN1:TAN1-YFP or pTAN1:CFP-TAN1) was transformed into the tan1 air9 double mutant, the phenotype was rescued such that plants looked similar to and grew as well as WT plants (Mir et al., 2018;Mills and Rasmussen, 2022).
TAN1 is an intrinsically disordered protein with no welldefined domains. It was divided into five conserved regions based on alignments of amino acid similarity across plant species. Region I, which covers the first approximately 130 amino acids of TAN1, is the most highly conserved and mediates TAN1 localization to the division site during telophase. This approximately 130 amino acid region also mediates interactions between TAN1 and POK1 in the yeast two-hybrid system (Rasmussen et al., 2011). When TAN1 missing the sequence for the first approximately 130 amino acids was transformed into the tan1 air9 double mutant, no rescue was observed (Mir et al., 2018). This suggests that the first approximately 130 amino acids of the TAN1 protein are critical for function in root growth and division plane positioning.
Here, we show that both AIR9 and TAN1 are required for POK1 to remain at the division site after PPB disassembly. We identified TAN1-POK1 interaction motifs within the first 132 amino acids using the yeast two-hybrid system. Interestingly, the first 132 amino acids of TAN1 (TAN1 1-132 ) are sufficient to rescue the tan1 air9 double mutant, but not when a TAN1-POK1 interaction motif was disrupted. We found that when full-length TAN1 with the same mutated motif was used, substantial rescue was observed, except for defects in phragmoplast guidance and loss of POK1 and TAN1 at the division site during metaphase and telophase. Together, this suggests that interactions between POK1 and AIR9, and TAN1 and POK1, as well as other yet unknown proteins, are important for division plane orientation and plant growth.

Results
Either TAN1 or AIR9 is sufficient to recruit and maintain POK1 at the division site To understand how known division site-localized proteins interact at the division site, we examined POK1 fused to YFP-POK1 (Lipka et al., 2014) localization in WT, tan1 air9 double mutant and single mutant Arabidopsis plants expressing the microtubule marker UBQ10:mScarlet-MAP4 (Pan et al., 2020). Our hypothesis was that POK1 localization would not be contingent on TAN1 or AIR9 and would therefore be unaltered in the tan1 air9 double mutant. In contrast to our hypothesis, YFP-POK1 was lost from the division site during metaphase and telophase and accumulated less frequently during preprophase and prophase. In WT cells, YFP-POK1 colocalized with PPBs in 71% of preprophase/prophase cells (n = 50/70 cells, 15 plants, Figure 1A) consistent with previous observations (Lipka et al., 2014;Schaefer et al., 2017). In the tan1 air9 double mutant cells, YFP-POK1 colocalized with 50% of PPBs during preprophase/prophase, which was not significantly different from the colocalization rate in WT cells (n = 27/54 cells, 15 plants, Figure 1, D and M; Fisher's exact test, P = 0.0165, not significant with Bonferroni correction). In WT cells, YFP-POK1 remained at the division site in all observed metaphase (n = 13/13 cells, Figure 1B) and telophase cells (n = 31/31 cells, Figure 1C), similar to previous studies (Lipka et al., 2014). In rare instances, YFP-POK1 also accumulated in the phragmoplast midline in WT cells (13%, n = 4/31, 11 plants, Figure 1M). In contrast, in tan1 air9 mutants, YFP-POK1 was lost from the division site in metaphase (n = 0/21 cells, Figure 1, E and M) and telophase (n = 0/44, Figure 1F). Interestingly, in tan1 air9 double mutant cells, although YFP-POK1 did not accumulate at the division site, it accumulated at the phragmoplast midline in 77% of cells (n = 34/44), which was a significantly greater midline accumulation frequency than the 13% observed in WT plants (n = 4/31 cells, Fisher's exact test, P 5 0.00001). Together, this shows that POK1 is not maintained at the division site after PPB disassembly and that instead it accumulates in the phragmoplast midline. We hypothesize that mislocalized phragmoplast midline accumulation of YFP-POK1 in the tan1 air9 mutant occurs because YFP-POK1 is not maintained at the division site.
Next, we examined YFP-POK1 localization in tan1 and air9 single mutants. YFP-POK1 localized to the division site during all mitotic stages, but aberrantly accumulated in the phragmoplast midline in the tan1 single mutant. Similar to in WT plants, YFP-POK1 colocalized with PPBs during preprophase or prophase in the tan1 mutant ( Figure 1G Figure 1I, n = 12/27), which is a significantly greater midline accumulation rate compared to that in WT plants (13%, n = 4/31, 10 plants, Fisher's exact test, P = 0.0094) or the air9 single mutant ( Figure 1L, 10%, n = 4/40). Aberrant phragmoplast midline accumulation of YFP-POK1 in the tan1 single mutant suggested that the POK1-TAN1 interaction might be required to maintain POK1 at the division site. This prompted us to examine their interaction more closely.
Amino acids 1-132 of TAN1 rescue the tan1 air9 double mutant POK1 interacts with both full-length TAN1 and the first 132 amino acids of TAN1 using the yeast two-hybrid system (Rasmussen et al., 2011). TAN1 missing the first 126 amino acids failed to rescue the tan1 air9 double mutant, suggesting that this part of the protein is critical for TAN1 function (Mir et al., 2018). To test the function of this region of the protein in Arabidopsis, the TAN1 coding sequence for the first 132 amino acids was fused to YFP (TAN1 1-132 -YFP) driven by the cauliflower mosaic p35S promoter and was then transformed into the tan1 air9 double mutant. We used p35S:TAN1-YFP in the tan1 air9 double mutant as our benchmark for rescue, as its ability to rescue the tan1 air9 double mutant was demonstrated previously (Mir et al., 2018). The progeny of several independent p35S:TAN1 1-132 -YFP lines rescued the tan1 air9 double mutant, as described in more detail below. Overall root patterning of tan1 air9 double mutants expressing either p35S:TAN1 1-132 -YFP or full-length p35S:TAN1-YFP was restored, while untransformed tan1 air9 double mutant roots had misoriented divisions ( Figure 2A; Supplemental Figure S1). Cell file rotation, which skews left and has large variance in the tan1 air9 double mutant ( Figure 2, B and C), was significantly rescued in both p35S:TAN1 1-132 -YFP and p35S:TAN1-YFP tan1 air9 lines (n = 37 and 41 plants, respectively), compared to the untransformed tan1 air9 control (Levene's test used due to nonnormal distribution, P 5 0.0001). Root length at 8 days after stratification was also restored ( Figure 2D). Interestingly, although TAN1 1-132 -YFP rarely co-localizes with PPBs in WT plants (Rasmussen et al., 2011) or in the tan1 air9 double mutant (10%, n = 9/89 cells, Figure 3A), PPB angles of p35S:TAN1 1-132 -YFP and p35S:TAN1-YFP tan1 air9 plants had significantly less variance compared to the untransformed control ( Figure 2E). Phragmoplast positioning defects of the tan1 air9 double mutant were also significantly rescued by p35S:TAN1 1-132 -YFP. Altogether, p35S:TAN1 1-132 -YFP rescued the phenotypes of the double mutant similar to full-length p35S:TAN1-YFP. This indicates that most functions that affect phenotypes assessed here are encoded by the first section of the TAN1 gene.
Disrupting TAN1-POK1 interaction alters TAN1 and POK1 localization to the division site and reduces tan1 air9 rescue To further understand how TAN1 functions, we disrupted its ability to interact with the kinesin POK1 using alanine scanning mutagenesis. Alanine scanning mutagenesis was used to replace six amino acids with six alanines across the first approximately 120 amino acids of TAN1 1-132 (described in the "Materials and methods"). After testing their interaction with POK1 using the yeast two-hybrid system, we identified seven constructs that lost interaction with POK1 (Supplemental Figure S2). Reasoning that highly conserved amino acids would be more likely to play critical roles in the TAN1-POK1 interaction, we selected TAN1 1-132 with alanine substitutions, replacing the highly conserved amino acids at positions 28-33 (INKVDK) with six alanines (TAN1(28-33A) 1-132 ) for analysis in Arabidopsis. Our hypothesis was that the mutated form of TAN1 1-132 (TAN1(28-33A) 1-132 ) would not rescue the tan1 air9 mutant due to lack of POK1 and TAN1 interaction. TAN1(28-33A) 1-132 was cloned into a plant transformation vector to generate p35S:TAN1(28-33A) 1-132 -YFP and transformed into the tan1 air9 double mutant. The p35S:TAN1(28-33A) 1-132 -YFP construct partially rescued the tan1 air9 double mutant ( Figure 4; Supplemental Figure S3). p35S:TAN1(28-33A) 1-132 -YFP in the tan1 air9 double mutant did not rescue cell file rotation defects (Figure 4, B and D) or phragmoplast angle defects ( Figure 4F). However, overall plant growth ( Figure 4C) and root length ( Figure 4E) showed intermediate rescue compared to unaltered p35S:TAN1 1-132 -YFP in the tan1 air9 double mutant. PPB angles in tan1 air9 double mutants expressing either p35S:TAN1(28-33A) 1-132 -YFP or p35S:TAN1 1-132 -YFP were similar, suggesting that the TAN1-POK1 interaction may not be required for PPB placement ( Figure 4F). These results suggest that the first 132 amino acids of TAN1 perform several vital functions, some of which are contingent or partially contingent on a likely interaction with POK1 in Arabidopsis.
To determine if full-length YFP-TAN1(28-33A) had reduced accumulation at the division site during telophase similar to TAN1(28-33A) 1-132 -YFP, fluorescence intensity levels were measured. During prophase, the YFP-TAN1(28-33A) fluorescence intensity at the division site compared to in the cytosol was comparable to the TAN1-YFP fluorescence intensity ratios. In contrast, the YFP-TAN1(28-33A) fluorescence intensity ratios during telophase were reduced to $1.6 compared with unaltered TAN1-YFP ($2.1) indicating that YFP-TAN1(28-33A) accumulated less at the division site during telophase (Supplemental Figure S6). Together, these data suggest that TAN1 is recruited to the division site during prophase without an interaction with POK1. Defects in phragmoplast positioning may be due specifically to the disruption of the TAN1-POK1 interaction in the tan1 air9 double mutant. Since AIR9 is missing, TAN1 and POK1 interactions may be more important. Moreover, phragmoplast guidance defects may be due to the lower accumulation of TAN1 at the division site that would normally be mediated by POK1 during telophase.

Discussion
In tan1 and air9 single mutants, POK1 localizes to the division site and there are no discernable division plane defects (Model in Supplemental Figure S7). However, in the tan1 air9 double mutant, POK1 co-localizes with the PPB but is lost from the division site during metaphase (Model in Figure 7). First, this suggests that TAN1 and AIR9 are not essential for POK1 co-localization with the PPB. Second, it suggests that POK1 is maintained at the division site after PPB disassembly via direct or indirect interactions with TAN1 or AIR9. We provide evidence that TAN1 interacts with POK1 through motifs within the first 132 amino acids of TAN1, as identified using the yeast two-hybrid system. Alignments of TAN1 proteins from representative monocots and dicots, such as Solanum lycopersium, Oryza sativa, Sorghum bicolor, Zea mays, and Brassica napus, showed that amino acids 28-33 (INKVDK) are highly conserved across plant species (Supplemental Figure S8). Amino acids 30-32 (VDK) are identical and the remaining residues within the motif have similar properties across these plant species. The high degree of conservation suggests that these amino acids are likely important for TAN1 function. When alanine substitutions of these amino acids were introduced into TAN1 and transformed into the Arabidopsis tan1 air9 double mutant, we observed reduced TAN1 and POK1 localization at the division site, as well as defects in phragmoplast positioning. Here, we hypothesize that amino acids 28-33 are essential for TAN1 and POK1 interaction in both the yeast twohybrid system and in Arabidopsis. In addition to several reports showing that TAN1 and POK1 interact using the yeast two-hybrid system (Müller et al., 2006;Rasmussen et al., 2011), bimolecular fluorescence complementation has also been used to show TAN1-POK1 interactions in Arabidopsis protoplasts (Lipka et al., 2014). Alanine substitutions at positions 28-33 of TAN1 may disrupt TAN1-POK1 interactions through misfolding that blocks the POK1 interaction site or by affecting the amino acids that directly . Maximum projections of three 1-mm Z-stacks. Scale bars = 10 mm. Some bleed-through from the mScarlet channel can be seen in the YFP-POK1 panels. A, YFP-POK1 and CFP-TAN1 frequently colocalized with the PPB. B, YFP-POK1 and CFP-TAN1 were maintained at the division site in metaphase. C, YFP-POK1 and CFP-TAN1 were maintained at the division site in all early telophase cells and late telophase. E, YFP-POK1 and CFP-TAN1(28-33A) colocalized with the PPB. F, YFP-POK1 and CFP-TAN1(28-33A) were maintained at the division site in most metaphase cells. CFP-TAN1(28-33A) was faint at the division site. G, Both YFP-POK1 and CFP-TAN1(28-33A) were observed at the division site in most early telophase cells. H, YFP-POK1 and CFP-TAN1(28-33A) were recruited to the division site in late telophase. I, Subcellular localization of CFP-TAN1 and CFP-TAN1(28-33A) in tan1 air9 double mutant cells during preprophase/prophase, metaphase, early telophase (telophase cells where the phragmoplast has not yet contacted the cell cortex), and late telophase. N 4 19 plants for each genotype. Statistically significant differences were determined using Fisher's exact test. Asterisks indicate significant differences in POK1 and TAN1/TAN1(28-33A) localization to the division site only. CFP-TAN1 and YFP-POK1 colocalized with the PPB more frequently (72%, 59/82 cells) than CFP-TAN1(28-33A) and YFP-POK1 (41%; 32/79 cells; P = 0.001). tan1 air9 plants expressing CFP-TAN1(28-33A) also had more PPBs that only accumulated YFP-POK1 (16%; 13/79 cells; P = 0.0461) compared to CFP-TAN1-expressing plants (6%, 5/82). In metaphase cells, CFP-TAN1 and YFP-POK1 were maintained at the division site in 100% of observed cells (13/13), compared to CFP-TAN1(28-33A) and YFP-POK1 which accumulated at the division site in 58% of cells (11/19, P = 0.0104). In early telophase, TAN1(28-33A) and YFP-POK1 accumulation at the division site was reduced (39%; 12/31 cells, P = 0.0001) compared to CFP-TAN1 and YFP-POK1 accumulation (100%, 14/14 cells). CFP-TAN1(28-33A)-expressing plants also accumulated both CFP-TAN1(28-33A) and YFP-POK1 at the division site and YFP-POK1 in the phragmoplast midline more frequently in early telophase cells compared to CFP-TAN1 expressing plants (26%; 8/31 cells; P = 0.0436). CFP-TAN1(28-33A) and YFP-POK1 were also absent at the division site and phragmoplast midline in a portion of early telophase cells (35%; 11/31 cells; P = 0.098). compared to CFP-TAN1 expressing plants (0/14 cells). CFP-TAN1(28-33A) and YFP-POK1 accumulated at the division site in 71% of late telophase cells (42/59 cells) compared to CFP-TAN1 and YFP-POK1 (60/63, P = 0.0004) Nineteen percent of late telophase cells accumulated both CFP-TAN1(28-33A) and YFP-POK1 at the division site and YFP-POK1 in the phragmoplast midline (11/59 cells) more frequently compared to CFP-TAN1-expressing plants (3/63 cells; P = 0.022). In some late telophase cells of TAN1(28-33A)-expressing plants CFP-TAN1(28-33A) accumulated at the division site alone (7%; 4/59 cells; P = 0.0518) or TAN1(28-33A) and YFP-POK1 failed to accumulate at the division site (3%; 2/59 cells; P = 0.2318) compared to CFP-TAN1-expressing plants where neither localization pattern was observed in late telophase (0/63 cells). mediate POK1 binding. Regardless of the exact mechanism(s) of POK1-TAN1 physical interactions or the possibility that yeast two-hybrid interactions do not reflect equivalent POK1-TAN1 physical interactions in Arabidopsis, we show that these TAN1 amino acids are involved in mediating TAN1 and POK1 localization to the division site.
We demonstrate that the first region of the TAN1 protein, the first 132 amino acids that primarily accumulate at the division site during telophase (Rasmussen et al., 2011), is both necessary (Mir et al., 2018) and sufficient to largely rescue the tan1 air9 double mutant (Figure 2). This suggests that TAN1 1-132 and its recruitment to the division site during telophase is critical for correct division plane orientation in the tan1 air9 double mutant. Although full length TAN1 localizes to the division site throughout cell division, the ability of TAN1 1-132 to rescue the tan1 air9 double mutant suggests that TAN1, and possibly POK1, localization to the PPB and division site during metaphase may not be required Figure 7 Speculative model of TAN1, AIR9, and POK1 interactions to ensure correct division plane orientation. A, In WT cells, AIR9, TAN1, and POK1 are recruited independently of one another to the PPB. Interaction between TAN1 and POK1 maintains both proteins at the division site through telophase, with AIR9 being re-recruited to the division site in late telophase. B, In the tan1 air9 double mutant, TAN1, AIR9, and potential AIR9/POK1 interacting proteins are recruited to the PPB. Upon disassembly of the PPB, POK1 is lost from the division site and during telophase aberrantly accumulates in the phragmoplast midline. Due to the loss of TAN1 and POK1 from the division site, the phragmoplast is not guided to the location defined by the PPB. C, In the tan1 air9 double mutant expressing TAN1(28-33A), TAN1(28-33A), and POK1 are recruited to the PPB independently of one another. POK1 and TAN1(28-33A) are partially maintained in some metaphase and early telophase cells possibly by interactions with other proteins. However, due to the inability of TAN1(28-33A) and POK1 to interact with one another, both proteins are not efficiently maintained at the division site. Most (90%) late telophase cells contain both POK1 and TAN1(28-33A) at the division site. Late recruitment of POK1 and TAN1(28-33A) may help guide the phragmoplast to the correct division site in most cells.
for division site maintenance in Arabidopsis. Indeed, whether the PPB itself is required for division plane positioning has been raised by analysis of a triple mutant in three closely related genes (TONNEAU RECRUITING MOTIF6 (TRM6), TRM7, and TRM8). The trm678 mutant, which lacks well defined PPBs, has partially disrupted POK1 recruitment to the division site but only minor defects in division positioning (Schaefer et al., 2017). However, when amino acids critical for TAN1-POK1 interactions in the yeast two-hybrid system are disrupted by transforming TAN1(28-33A) 1-132 -YFP into the tan1 air9 double mutant, root growth and phragmoplast positioning are disrupted. TAN1(28-33A) 1-132 -YFP accumulation at the division site during telophase was reduced compared to unaltered TAN1 1-132 -YFP. This suggests that TAN1-POK1 interaction promotes, but is not strictly necessary, for TAN1 recruitment to the division site during telophase.
Full-length TAN1(28-33A) localizes to the division site throughout cell division and almost fully rescues the tan1 air9 double mutant. TAN1, AIR9, and POK1 colocalize at the PPB independently of one another, which may promote the formation of protein complexes required for division site maintenance. Colocalizing with the PPB may provide an opportunity for nearby proteins to form stabilizing interactions before PPB disassembly. This suggests that recruitment of TAN1 and POK1 to the division site early in cell division may provide another temporally distinct way to promote correct division plane positioning. Phragmoplast positioning defects in TAN1(28-33A) tan1 air9 plants may be the result of defects in phragmoplast guidance in cells that lacked TAN1(28-33A) and POK1 at the division site in metaphase or early telophase that were not corrected in late telophase.
The ability of TAN1(28-33A) and POK1 to remain at the division site in some cells after PPB disassembly in the tan1 air9 double mutant suggests that there are other proteins that interact with TAN1 and/or POK1 that help stabilize them at the division site perhaps via the formation of multiprotein complexes. The pleckstrin homology GAPs, PHGAP1, and PHGAP2 (Stöckle et al., 2016); RANGAP1 (Xu et al., 2008); and IQ67 DOMAIN (IQD)6,7,8 proteins (Kumari et al., 2021) are division site-localized proteins that may stabilize TAN1 and POK1 at the division site via their interaction with POK1. PHGAP1, PHGAP2, and RANGAP1 are dependent on POK1 and POK2 for division site recruitment. Like TAN1, RANGAP1 colocalizes with the PPB and remains at the division site throughout cell division (Xu et al., 2008). PHGAP1 and PHGAP2 are uniformly distributed in the cytoplasm and on the plasma membrane in interphase cells and accumulate at the division site during metaphase. These proteins also have their own distinct roles in division site maintenance (Stöckle et al., 2016). PHGAP2 has a likely role in division site establishment by regulating ROP activity (Hwang et al., 2008). RANGAP1 regulation of local RAN-GTP levels has potential roles in microtubule organization and division site identity (Xu et al., 2008). IQD6, IQD7, and IQD8 interact with POK1 and play a role in PPB formation and POK1 recruitment to the division site. The iqd678 triple mutant has PPB formation defects and fails to recruit POK1 to the division site in cells lacking PPBs. However, POK1 localization to the division site in the iqd678 mutant recovers during telophase to WT levels (Kumari et al., 2021). We speculate that this IQD6-8 independent recruitment may depend on TAN1. Unlike the PHGAPs and RANGAP1, IQD8 localization to the division site is not dependent on POK1 and POK2. This suggests that IQD6-8 proteins work upstream of POK1 to establish the division site and are important for POK1 recruitment to the division site early in cell division. Although TAN1-POK1 interactions become critical for TAN1 and POK1 maintenance at the division site in the absence of AIR9, other division site-localized proteins may provide additional stability and help maintain TAN1 and POK1 at the division site.
How AIR9 stabilizes POK1 at the division site in the absence of TAN1 is less clear. There is no information about whether POK1 and AIR9 interact directly with one another. Additionally, AIR9 localization, in contrast to TAN1 localization, is intermittent at the division site. When expressed in tobacco (Nicotiana tabacum) Bright Yellow2 cells, AIR9 colocalizes with the PPB but is then lost from the division site until late telophase when the phragmoplast contacts the cortex (Buschmann et al., 2006). In Arabidopsis, AIR9 may localize to the division site during metaphase or telophase, but it is difficult to observe because AIR9 also strongly colocalizes with cortical microtubules, which may obscure AIR9 localization in nearby cells (Buschmann et al., 2015). Rather than directly interacting with POK1, AIR9 may recruit other proteins to the division site during preprophase that help maintain POK1 at the division site in the absence of TAN1. One potential candidate is the kinesin-like calmodulin binding protein, KCBP, which interacts with AIR9 (Buschmann et al., 2015). KCBP is a minusend-directed kinesin (Song et al., 1997) that localizes to the division site in Arabidopsis and moss (Miki et al., 2014;Buschmann et al., 2015). We speculate that other TAN1, AIR9, and POK1 interacting proteins that have not been identified yet may be key for TAN1-POK1 division site maintenance.
POK1 and POK2 have roles in phragmoplast guidance, but POK2 also has a role in phragmoplast dynamics (Lipka et al., 2014;Herrmann et al., 2018). Although POK1 does not frequently accumulate in the phragmoplast midline in WT cells, POK2 showed striking dual localization to both the phragmoplast midline and the division site. Localization of POK2 to the phragmoplast midline required the N-terminal motor domain, while the C-terminal region was localized to the division site . Our hypothesis is that the "default" location of both POK1 and POK2 is at microtubule plus ends at the phragmoplast midline, based on likely or confirmed plus end-directed motor activity (Chugh et al., 2018). Interactions with division site-localized proteins, such as TAN1 and AIR9, may stabilize or recruit POK1 and POK2 at the division site away from the phragmoplast midline.
We demonstrate that AIR9 and TAN1 function redundantly to maintain POK1 at the division site to ensure correct cell wall placement. In the absence of AIR9, our data suggest that TAN1-POK1 interaction promotes, but is not required for, the maintenance of both proteins at the division site, and disrupting this interaction partially disrupts their localization to the division site. This also suggests that other TAN1, POK1, and AIR9 interacting proteins are involved with stabilizing TAN1 and POK1 at the division site.

Materials and methods
Growth conditions, genotyping mutants, and root length measurements Arabidopsis (A. thaliana) seedlings were grown on 1/2 strength Murashige and Skoog (MS) media (MP Biomedicals; Murashige and Skoog, 1962) containing 0.5 g L -1 morpholineethanesulfonic acid monohydrate (MES-monohydrate, Fisher Scientific, Waltham, MA, USA, CAS 145224-94-8), pH 5.7, and 0.8% agar (Fisher Scientific). Seeds sown on plates were first stratified in the dark at 4 C for 2-5 days, then grown vertically in a growth chamber (Percival) with 16-h white light $111 mE*m -2 s -1 (F17T8/TL741 Fluorescent Tube (Philips)/8-h dark cycles and the temperature set to 22 C. For root length experiments, tan1 air9 transgenic T3 lines expressing p35S:TAN1-YFP, p35S:TAN1 1-132 -YFP, p35S: TAN1(28-33A) 1-132 -YFP, or p35S:YFP-TAN1(28-33A) were grown vertically, the plates were scanned (Epson) and root lengths were measured using FIJI (ImageJ, http://fiji.sc/) after 8 days. Untransformed tan1 air9 double mutant and air9 single mutant seeds were grown alongside the double mutant seeds expressing the TAN1 constructs in equal numbers on the same plates to ensure plants were grown under the same conditions. After plates were scanned, seedlings were screened by confocal microscopy to identify seedlings expressing YFP translational fusion transgenes and CFP-TUBULIN, if present in the transgenic lines. At least three biological replicates, grown on separate plates on separate days, and at least 28 plants of each genotype across all replicates were analyzed for each root growth experiment. Welch's t tests were used to identify whether there were statistically significant differences between replicates before pooling the replicates for analysis. Root lengths were then plotted using Prism (GraphPad). Statistical analysis of root length was performed with Prism (GraphPad) using t tests with Welch's correction. Welch's t test (unequal variance t test) is used to test the hypothesis that two populations have equal means. Unlike the Student's t test, Welch's t test is often used when two samples have unequal variances or sample sizes. This test was used due to the unequal sample sizes because the plants examined were often segregating for multiple transgenes and had lower sample sizes than control plants such as the air9 single mutant and tan1 air9 double mutant, which either lacked transgenes or were segregating fewer transgenes.

Microscopy
An inverted Ti Eclipse (Nikon, Tokyo, Japan) with motorized stage (ASI Piezo) and spinning-disk confocal microscope (Yokogawa W1) built by Solamere Technology was used with Micromanager software (micromanager.org). Solid-state lasers (Obis) and emission filters (Chroma Technology) were used. For CFP translational fusions, an excitation of 445 and emission of 480/40 were used; for YFP translational fusions, an excitation of 514 and emission of 540/30 were used; and for propidium iodide (PI), Alexa-568 goat anti-mouse antibody, and mScarlet-MAP4, an excitation of 561 and emission of 620/60 were used. A 20Â objective with a 0.75 numerical aperture and a 60Â objective with a 1.2 numerical aperture were used with perfluorocarbon immersion liquid (RIAAA-6788 Cargille). Excitation spectra for mScarlet-MAP4 and YFP-POK1 partially overlapped, which resulted in a faint bleed-through signal in the YFP channel for some dense microtubule structures (e.g. spindles and phragmoplasts). YFP-POK1 colocalization with PPBs was carefully determined based on distinct YFP-POK1 signal and the presence of cytosolic YFP-POK1.
The ratio of the division site versus cytosolic fluorescence intensity was determined by taking the median YFP fluorescence intensity from the center Z-stack of individual cells with PPBs or phragmoplasts. For each cell, the median fluorescence intensity was measured for two cytosolic areas and the division site on each side of the cell using circles with areas of 0.875 lm 2 . The sum of the median intensity at the division site on each side was then divided by the sum of the median intensity of the two cytosolic areas to calculate the ratio of the division site versus cytosolic fluorescence intensity. Fluorescence intensities were measured in FIJI. All plants used for this analysis were grown on the same day and imaged using identical conditions, and at least five plants of each genotype were examined.

Measurements of PPB and phragmoplast angles and cell file rotation
At least three biological replicates, grown on separate plates on separate days, composed of at least 15 plants per genotype for PPB measurements and at least eight plants per genotype for phragmoplast measurements were used to gather angle data. Eight-day-old seedlings were stained with 10-lM PI for 1 min and then destained in distilled water before imaging by confocal microscopy using a 20Â or 60Â objective. PPB and phragmoplast angles were measured using FIJI. The angle was measured between the left-hand cell wall and the orientation of the PPB or phragmoplast in the root tips of tan1 air9 double mutant plants expressing CFP-TUBULIN or immunostained microtubules (described in the next section). Cell file rotation was examined by measuring from the left-hand side of the transverse cell wall relative to the long axis of the root in images of the differentiation zone stained with PI. The differentiation zone was identified by the presence of root hairs. Prism (GraphPad) and Excel (Microsoft Office) were used to perform statistical analyses and to plot data. F-tests were used to compare normally distributed variances (PPB and phragmoplast angles) and Levene's tests were used to compare nonnormally distributed variances (cell file rotation angle measurements). The tan1 air9 double mutant has nonnormally distributed cell file twisting because the roots tend to twist to the left (Mir et al., 2018). Genotypes across biological replicates were compared to ensure there were no statistically significant differences between them before pooling data.

Yeast two-hybrid
Six alanine substitutions were generated using the overlapping PCR and TAN1 coding sequence in pEZRK-LNY-TAN1 1-132 -YFP as a template beginning at amino acid 10 of TAN1 and continuing through to amino acid 123 according to Russell and Sambrook (2001). All amino acids except substitutions for 64-69 and 106-111 were cloned into the pAS vector (Fan et al., 1997) using EcoRI-BamHI double digestion. pBD-TAN1(28-33A) was generated using primers Ala_05_FOR and Ala_05_REV to perform DpnI-mediated site-directed mutagenesis by PCR (Fisher and Pei, 1997). pBD-TAN1 (Walker et al., 2007) and pAS-TAN1 1-132 (Rasmussen et al., 2011) were used as positive controls, while pAD-MUT was used as a negative control for testing interactions with pAD-POK1 (Müller et al., 2006). pAD-POK1 and pAS-TAN1 1-132 constructs were co-transformed into yeast strain YRG2 according to manufacturer instructions (Stragene). A positive yeast two-hybrid interaction was determined by the presence of growth on plates cultured at 30 C lacking histidine after 3 days. Plates were then scanned (Epson, Nagano, Japan).

Supplemental data
The following materials are available in the online version of this article.
Supplemental Figure S7. A model of POK1 localization in tan1 and air9 single mutants.
Supplemental Figure S8. Alignments of amino acids 1-55 of A. thaliana TAN1 with TAN1 homologs from other plant species.
Supplemental Table S1. Primers used for cloning and genotyping.
Supplemental Data Set 1. Statistical analysis of tables.